Research Summary: Research in the Molecular Pathogenesis Section is focused on defining changes in the genes that underlie inherited susceptibilities to common diseases such as cancer and birth defects. Changes in folate and vitamin B12 metabolism are associated with tumor formation, birth defects and cognitive decline. Folate and vitamin B12 genes are also involved in the methylation of DNA and normal brain function. We are searching for genetic variants in genes related to folate, methionine and homocysteine metabolism. Individuals affected with spina bifida (one form of neural tube defects) will be tested for these variants. Variants found at higher frequency in individuals with disease will help us identify genes associated with risk. Our most recent report (VanderMeer 2016) evaluated genetic variation in the receptor responsible for dietary folate uptake. We observed suggestive but not conclusive evidence of association with neural tube defects and oral clefting defects. The field of neural tube defects has historically focused on evaluating variation in genes in the folate/vitamin B12 metabolic pathway. As genotyping technology has increasingly offered more information at lower cost, the next logical step in this research is to screen the entire genome for additional genes associated with NTDs. This type of experiment requires a very large sample size. Although we have one of the worlds largest samples of NTDs that are available for genetic research, our sample is too small to carry out a genome wide association study (GWAS). In collaboration with Anne Molloy, Trinity College Dublin, we have organized and international collaboration with the goal of pooling samples for a GWAS. Over ten groups have joined this collaborative effort. The total number of samples collected by all groups exceeds 5,000. We have obtained external funding to coordinate this study and collect the samples at a central location. We have collected DNA samples from these collaborators, and in the past year we have continued recruitment of additional investigator participation in replication studies. Other groups have measured the impact of genetic variants on the level of vitamin B12 in blood. We have measured vitamin B12 in the blood of over 5,000 individuals using standard methods. We then subjected these samples to assays that resolve circulating vitamin B12 into pools that correspond to its two major carrier proteins; transcobalamin, the bioactive carrier that is taken up by all cells, or haptocorrin, which is taken up by the liver for eventual recirculation or elimination. We currently have a manuscript in review describing our insight that the variant repeatedly reported to influence circulating vitamin B12 is actually influencing the subset of vitamin B12 bound to haptocorrin. In a second study of this cohort (Molloy 2016), we published in the past year that the common genetic variant most associated with a circulating marker of vitamin B12 deficiency was in a gene unrelated to vitamin B12 transport or metabolism. These findings could be relevant to clinical measures of vitamin B12 testing where it is known that individuals on either side of the normal range are given false positive and negative results. It may be possible to use individual genetic variation to refine the interpretation of clinical testing. Related to this, we also recently published our work (Sobczyska-Malefora, 2016) on the genetic underpinning of two individuals who showed an absence of the bioactive vitamin B12 carrier, transcobalamin. We found that these individuals both have a genetic variant that apparently interferes with the way transcobalamin is detected in the assay used, and they in fact have normal transcobalamin able to carry vitamin B12. Our work highlights how factoring in the influence of genetic variation will be essential to accuracy in diagnostics. We are also using animal models to understand the biology of genes involved in vitamin B12 metabolism. We have developed strains of zebrafish and mice in which we have disrupted vitamin B12 transport genes. In our zebrafish model, we targeted the only known circulatory carrier of vitamin B12. Although these fish should not be able to deliver vitamin B12 to their cells and tissue, they appear to develop normally. This led to a search for an alternate vitamin B12 transport protein in zebrafish. A bioinformatics approach revealed two coding regions that are highly similar to the known carrier protein. We have shown that in an artificial system these partial proteins can be expressed and bind vitamin B12. We are in the process of determining whether these proteins may have a biological role in vitamin B12 transport in these fish. Our other model of vitamin B12 deficiency is in mice, where we have targeted the cellular receptor for vitamin B12 uptake. These animals appear to mimic a number of aspects of vitamin B12 deficiency in humans, especially when placed on a diet lacking vitamin B12. First, these mice exhibit the metabolic hallmarks of vitamin B12 deficiency observed in humans (elevated circulating homocysteine and methylmalonic acid). They are also prone to developing anemia as they age, which can be temporarily rescued with an injection of vitamin B12. Last, we have been investigating female-specific infertility in these mice. These dams appear to ovulate normally, and we have shown their embryos can develop for a few days but implantation is generally unsuccessful. Future work is needed to determine whether vitamin B12 deficiency in the offspring also contributes to the apparent infertility of their dams. Future work with this animal model may include exploring the impact of vitamin B12 deficiency on neurological function (gene expression in the brain), cell division (e.g., melanocyte activity as it relates to hair color), and retinal health. The literature contains a variety of strength of evidence of the impact of vitamin B12 on these aspects of human health, and our mouse model provides a way to more fully interrogate these processes. Over the past year we have carried out studies aimed to find the genetic bases of metabolic traits related to folate, vitamin B12 and one carbon metabolism.